Measurement of the Thermal Unfolding of Proteins by Differential Scanning Calorimetry and Fluorescent Reporter Dyes.
Brian E. Lang, Kenneth D. Cole
Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) were used to measure the unfolding of two monoclonal antibodies and several widely available proteins to compare and contrast the two techniques. While the DSF technique has been shown to have some correlation with melting temperature (Tm) determined by DSC for a limited number of proteins and fluorescent dyes, there is little data to compare the two techniques with different types of proteins and dye combinations. The effect of the temperature scan rate on the observed transition temperatures for both techniques was measured in this study, since protein unfolding is often influenced by kinetic effects. We determined the melting temperature of the proteins using DSC over a range of temperature scan rates and compared them to the transition temperatures from using DSF over comparable temperature scan rates. Commonly used fluorescent dyes and five proteins, including the NISTmAb (soon to be available as a monoclonal antibody reference material) were used. We also compared the calculation of the Tm from DSF experiments by using either the midpoint of the fluorescent signal or from the peak values of the curves. Generally the peak values had a better correlation than the midpoint values when compared to the values determined from DSC. We have shown that the scan rate is an important variable when comparing results between DSC and DSF.
protein unfolding, thermodynamics, differential scanning calorimetry, differential scanning fluorimetry, monoclonal antibodies, RNase A, invertase